WEE 1 is a key eukaryotic cell cycle regulator. In plants it has a clear role at the DNA damage/ DNA replication checkpoints. I aimed to discover the functional significance of interactions between WEE1 and other cellular proteins in Arabidopsis thaliana and Nicotiana tabacum. First I examined effects of ectopic expression of Arabidopsis WEE1 (Arath;WEE1) in transgenic tobacco and tobacco WEE1 (Nicta;WEE1) in transgenic Arabidopsis. Western blotting using a plant WEE1 antibody showed that expression of Nicta;WEE1 in Arabidopsis caused increases in total WEE1 protein. The response of primary root length, numbers of lateral roots and primordia, and meristem length to zeocin (a DNA damaging agent) and hydroxyurea, (which perturbs DNA replication), resembled the wee1-1 insertional mutant rather than Arath;WEE1 over-expression. Expression of Arath;WEE1 in tobacco resulted in reduced WEE1 protein but also induced similar phenotypic changes as Nicta;WEE1 expression in Arabidopsis under zeocin and HU stress. I concluded that interactions with cellular proteins in the alien species resulted in down-regulation of WEE1 activity. In a yeast 2-hybrid screen Arath;WEE1 interacted with the glutathione-S-transferase protein, GSTF9. To test the functionality of this interaction I analysised the root and cell cycle phase phenotypes of single mutants: wee1-1 and gstf9 and I generated the double mutant wee1-1;gstf9. I demonstrated that both Arath;WEE1 and GSTF9 have roles in the DNA replication and damage checkpoints, but largely act in different genetic pathways. Arath;WEE1 also interacts with GF14ω, a 14-3-3 protein in a yeast 2-hybrid assay. In other eukaryotes this stabilizes WEE1. I confirmed that over-expression of GFF14ω in transgenic Arabidopsis (GFF14ω OEX) results in a very similar root phenotype to over-expression of Arath;WEE1 as predicted from a stabilization of WEE1. However the GFF14ω OEX phenotype was not abolished in a wee1-1 genetics background.indicating that Arath;WEE1is not required for the action of GF14ω.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:567308 |
| Date | January 2012 |
| Creators | Rafiei, Golnaz |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/30683/ |
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